First Polyphasic Study of Cheffia Reservoir (Algeria) Cyanobacteria Isolates Reveals Toxic Picocyanobacteria Genotype

Monitoring water supply requires, among other quality indicators, the identification of the cyanobacteria community and taking into account their potential impact in terms of water quality. In this work, cyanobacteria strains were isolated from the Cheffia Reservoir and identified based on morphological features, the 16S rRNA gene, phylogenetic analysis, and toxin production by polymerase chain reaction PCR screening of the genes involved in the biosynthesis of cyanotoxins (mcyA, mcyE, sxtA, sxtG, sxtI, cyrJ, and anaC). Thirteen strains representing six different genera: Aphanothece, Microcystis, Geitlerinema, Lyngbya, Microcoleus, and Pseudanabaena were obtained. The results demonstrated the importance of morphological features in determining the genus or the species when incongruence between the morphological and phylogenetic analysis occurs and only the utility of the 16S rRNA gene in determining higher taxonomic levels. The phylogenetic analysis confirmed the polyphyly of cyanobacteria for the Microcystis and Oscillatoriales genera. Unexpectedly, Aphanothece sp. CR 11 had the genetic potential to produce microcystins. Our study gives new insight into species with picoplanktonic (or small) cell size and potentially toxic genotypes in this ecosystem. Thus, conventional water treatment methods in this ecosystem have to be adapted, indicating the requirement for pre-treatment methods that can effectively eliminate picocyanobacteria while preserving cell integrity to prevent toxin release.


Introduction
Cyanobacteria are photosynthetic prokaryotes found in aquatic and terrestrial habitats [1].These organisms are prolific producers of natural products recognized as toxins with hepatotoxic, neurotoxic, and dermatotoxic effects and are harmful to humans and animals [2].For people, the Caruraru syndrome that occurred in Brazil (Caruaru City) was the most severe event.During this incident, 76 patients died in a hemodialysis clinic; analysis of the liver tissue and serum of the victims led to the identification of microcystins (MCs, hepatotoxic cyanotoxins) [3].Nowadays, the monitoring of water intended for public use takes account of the identification of toxic cyanobacteria [4].Moreover, the elimination of cyanobacteria in drinking water treatment services depends on the cyanobacterial species and the treatment procedures employed [5].For instance, Aphanezomenon cells exhibit a higher propensity to traverse traditional sand filtration systems, leading to the discharge of intracellular toxins into treated drinking water [6].
Morphological analyses have been the basis of cyanobacterial classification systems [7].However, because of limitations due to the extreme phenotypic flexibility depending on different environmental and culture conditions [8], a polyphasic approach combining morphological and molecular information has been used for accurate cyanobacteria identification [9].Nevertheless, this approach cannot differentiate between toxic and nontoxic cyanobacteria strains.Accordingly, various immunological and analytical approaches, such as enzyme-linked immunosorbent assays (ELISA), the protein phosphatase inhibition (PPI) assay, chromatography, and mass spectrometry [4], have been used for this objective.However, sensitivity, specificity, and high-cost limitations have made them inadequate for routine use [10].The capacity of cyanobacteria to produce toxins is associated with specific metabolic processes that are encoded by complex gene operons.Toxin-related genes PCR amplification is a reliable technique for detecting potential toxic strains [11].
In Algeria, several reports have described the occurrence of cyanobacteria in ecosystems with socio-economic importance [12][13][14].The Cheffia Reservoir is a water body in northeastern Algeria; the water is used for drinking and irrigation.Previous reports have been limited to analyzing environmental samples and toxins in this ecosystem and identifying cyanobacteria by a culture-independent technique [12].The cultivation approach has advantages over the cultivation-independent studies of cyanobacteria; it allows for detailed phenotypic, genetic, physiological, and biochemical characterization [15].Genetic research with axenic cultures is critical for cyanobacteria polyphasic taxonomy investigations, particularly for understudied polyphyletic taxa [16].Recent studies have shown that numerous newly described cyanobacteria are exclusively based on isolated strains [17][18][19][20].Thus, the aim of this study was focused specifically on cyanobacterial isolates from the Cheffia Reservoir.Isolated strains were identified using morphological features and phylogenetic analysis.Furthermore, toxin-related genes were localized using specific primers.

Collection of Cyanobacterial Samples and Isolation
The cyanobacteria sample was collected from the Cheffia Reservoir (36 • 36 33.5 N 8 • 02 34.8 E, North-East of Algeria, Figure 1) using a plankton net (20 µm mesh size) on 21 October 2018.Cyanobacteria isolation was done by the enrichment of cyanobacterial environmental samples in a liquid culture medium, then plating by streaking each sample on a 1.5% agar culture medium.The plates were observed regularly under an inverted microscope Leica ® DMi8 (Leica Microsystems, Wetzlar, Germany), and isolated strains were transferred to new plates and also maintained on a liquid medium.The cyanobacteria were isolated, grown, and maintained in BG11 medium [21] under a 14:10 h light/dark cycle at 19 ± 1 • C. Cycloheximide (Sigma, Dorse, UK) at 100 µg mL −1 was added to the culture medium in the first isolation steps to inhibit the growth of eukaryotic microorganisms [22].

Morphological Characterization
Morphological studies of isolated strains were carried out under an Olympus BX51 light microscope at 100×, 400×, and 1000× magnifications.In addition, photographs were taken with a Leica ® DFC550 digital camera, and measurements were determined for each strain (n = 100 cells) at 1000× magnification using Leica ® LAS X V.4 software.The strains were classified according to the morphological criteria of Komárek and Anagnostidis [24,25].

Morphological Characterization
Morphological studies of isolated strains were carried out under an Olympus BX51 light microscope at 100×, 400×, and 1000× magnifications.In addition, photographs were taken with a Leica ® DFC550 digital camera, and measurements were determined for each strain (n = 100 cells) at 1000× magnification using Leica ® LAS X V.4 software.The strains were classified according to the morphological criteria of Komárek and Anagnostidis [24,25].
For the obtained sequences with low DNA quality, purified PCR products were cloned into the pCR™2.1-TOPO® vector (Invitrogen TOPO ® TA Cloning Kit) and then transformed into Escherichia coli OneShot ® TOP10 cells (Invitrogen) following the instructions of the manufacturers.After the white-blue selection on the ampicillin 1.5% agarose plates with Luria Bertani (LB) medium, the colonies were transferred into fresh liquid LB medium with 100 µg ml -1 of ampicillin and cultured overnight at 37 • C with shaking at 2000 rpm.The Plasmid DNA was isolated using GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions.The plasmids were sequenced bidirectionally, using the following primers: 27F1, CYA359F, CYA781R, and 1494Rc, by Sanger sequencing at GATC Biotech (Ebersberg, Germany).

Sequence Analysis
Raw forward and reverse sequences were assembled using Geneious (version 8.1.8,Biomatters Ltd., Auckland, New Zealand) and then deposited in the GenBank database "http://www.ncbi.nlm.nih.gov/(accessed on 26 January 2022)" under the accession numbers OM401262 to OM401274.The obtained sequences were compared to previously published sequences in NCBI (National Center for Biotechnology Information; accessible via http://www.ncbi.nlm.nih.gov/) using the BLASTN algorithm.Based on the resulting blast analysis, a phylogenetic tree was constructed using the "One Click" mode of the online web service Phylogeny.frplatform (http://www.phylogeny.fr/simple_phylogeny.cgi [28]).

Phylogenetic Study
BLAST analysis showed high sequence similarities ranging from 97.26 to 99.91% between the 16S rRNA gene sequences of cyanobacterial isolates from the Cheffia Reservoir and 27 strain sequences from the Chroococcales, Oscillatoriales, and Synechococcales orders accessible in GenBank (Table 4).The phylogenetic tree obtained with the studied strains is shown in Figure 4.The tree included five main clusters.Cluster A comprised sequences of Microcoleus sp. and Lyngbya spp.Sub-cluster I included Microcoleus sp.CR8 isolate and Microcoleus HTT-U-KK5 strain deposited in GenBank.Sub-cluster II contained Lyngbya isolates with sequences from the same genera: L. martensiana H3b/33 and two other sequences from Oscillatoria UIC 10045 and Phormidium cf.irriguum CCALA 759.Cluster B consists only of the Microcystis flos-aquae CR13 isolated in this work with sequences from the same species, M. flos-aquae CHAB541, and those of Synechocystis SAG 45.90 and Sphaerovacum brasiliense CCIBt3094, from freshwater eutrophic reservoirs and seawater from Europe, Asia, and South America, deposited in GenBank.Cluster C is represented by G. amphibium strains with the strain G. amphibium and Limnothrix, Jaaginema, Pseudanabaena, and Anagnostidinema sequences.Cluster D included the two strains of the picocyanobacterial genera Aphanothece and the isolate identified morphologically as Microcystis sp.grouped with sequences belonging to Cyanobacterium, Cyanobium, Synechococcus, Aphanothece, Cyanodictyon, Aphanocapsa, and Microcystis deposited in GenBank.Finally, cluster E included the Psedanabaena isolate and sequences in the GenBank database of the terrestrial and freshwater species: P. limnetica Lim1, L. redekei CCAP 1459/29 and Arthronema gygaxiana UTCC 393 isolated from France, Japan, and Italy.

PCR Amplification of Toxin-Encoding Genes
The molecular screening of the genes involved in the biosynthesis of microcystin was positive for the mcyE-specific microcystin/nodularin gene for Aphanothece sp.CR11 strain, as shown in Figure 5.However, none of the isolated strains tested positive for anatoxin (anaC), saxitoxin (sxtA, sxtG, and sxtI), or cylindrospermopsin (cyrJ) genes.

PCR Amplification of Toxin-Encoding Genes
The molecular screening of the genes involved in the biosynthesis of microcystin was positive for the mcyE-specific microcystin/nodularin gene for Aphanothece sp.CR11 strain, as shown in Figure 5.However, none of the isolated strains tested positive for anatoxin (anaC), saxitoxin (sxtA, sxtG, and sxtI), or cylindrospermopsin (cyrJ) genes.

PCR Amplification of Toxin-Encoding Genes
The molecular screening of the genes involved in the biosynthesis of microcystin was positive for the mcyE-specific microcystin/nodularin gene for Aphanothece sp.CR11 strain, as shown in Figure 5.However, none of the isolated strains tested positive for anatoxin (anaC), saxitoxin (sxtA, sxtG, and sxtI), or cylindrospermopsin (cyrJ) genes.

Discussion
In order to characterize cyanobacteria isolates from the Cheffia Reservoir, morphological, molecular and phylogenetic analyses were conducted.The morphological identification up to the species level was successful for some strains.More importantly, the strains from the genera Geitlerinema, Lyngbya and Microcystis showed substantial phenotypic plasticity.All the isolated Geitlerinema strains have identical morphological characteristics and are similar to G. amphibium described by Bittencourt-Oliveira et al. [36].However, our isolates vary from these organisms; their cell length was less than these organisms, which are 2.2-7 µm long, and their cell width was relatively wider than these organisms, which are 1.0-2.2µm wide.The cells of isolated strains belonging to the species P. rosea, L. nigra, L. cincinnata, and L. stagnina exhibited a larger width than those described by Komárek and Anagnostidis [24], which are 1.61-2.67,12.82-17.84,13.46-19.66and 13.00-19.36µm, respectively.As well, the M. flos-aquae strain had cells with a larger diameter than the M. flos-aquae described by Komárek and Anagnostidis [25], which is 3.5-4.8µm.The genus Microcystis has previously shown significant morphological flexibility [37][38][39].This morphological plasticity has been related to several environmental or cultivation factors, such as medium composition, temperature, and light intensity [40,41].
Our findings also showed that morphological and phylogenetic classifications might be incompatible.This is the case of the strain Microcystis sp.CR 12, assigned to the genus Microcystis based on the morphological description of this genus in Bergey's Manual of Determinative Bacteriology [42].The genus Microcystis is characterized by the following features: coccoid cells with aerotopes and a tendency to form colonies delimited by mucilage.Differences between molecular and morphological descriptions have been extensively reported [43][44][45][46].Moreover, it has also been demonstrated that GenBank contains erroneously classified species.Strains in culture collections have also been misnamed, and they may be found in GenBank and other culture collections under various names [43].Moreover, with the numerous revisions to cyanobacteria taxonomy, there is no simple way to update existing databases [47], generating even more difficulty for cyanobacteria assignment [30,44].Nonetheless, the number of 16S rRNA gene sequences now accessible in global databases for some species is still low, and additional systematic efforts will be necessary to clarify existing taxonomic ambiguities [30].
The isolated Lyngbya strains were morphologically assigned to three distinct species, although phylogenetically related to one species, L. martensiana H3b/33.The reliability of 16S rRNA gene sequences to identify Lyngbya strains at the species level and lower has been contested [48].Engene et al. [49] suggested only using 16S rRNA gene sequences to identify Lyngbya strains at the genus level.Furthermore, Fathalli et al. [50] confirmed that even when closely related species are morphologically different, it may not be possible to differentiate them properly by the 16S rRNA gene.The 16S rRNA gene is less effective for phylogenetic investigations of closely related organisms due to its conserved character and lower evolutionary rate fluctuation than protein-encoding genes [51].
Three distinct clades were obtained from the Oscillatoriales strains, confirming the polyphyletic origin of this order [52].The obtained Microcystis morphospecies were mixed in the phylogenetic tree and cannot be identified as monophyletic organisms, which also confirms the polyphyly of the Chroococcales [16,53].
Our study represents the first report of a potentially toxic genotype from the genus Aphanothece in this ecosystem.Picocyanobacteria MCs producers have been isolated from the Tabocas reservoir in the city of Caruaru, where the first human poisoning episode was reported [54].MCs are the most dangerous group of cyanotoxins; they inhibit serine and threonine phosphatases-PP1 and PP2A-causing significant hepatotoxicity and acting as a carcinogen [55].
Some studies have also shown that strains of picocyanobacteria can produce MCs in culture, but no study has explored the presence of toxicity genes [56,57].The picocyanobacteria group can also produce blooms responsible for the significant loss of benthic wildlife [57][58][59][60].The most studied picocyanobacteria blooms have been observed in the northern Mediterranean at the Comacchio lagoons (northwest Adriatic coast) [61].These blooms led to the death of bottom flora and benthic fauna and the loss of valuable resources of fish (eel and mullet) and mollusks [62].Śliwi ńska-Wilczewska et al. [57] and Felpeto et al. [63] suggest that picocyanobacteria blooms are a new phenomenon that requires comprehensive studies.Although the impacts of climate change on picocyanobacterial blooms are diverse, most current evidence suggests that this process increases the amplitude and frequency of these events [57,64].So far, little is known regarding the toxicity of picocyanobacteria, whereas the number of reports about their prevalence in ecosystems is growing.Therefore, the problem of picocyanobacteria toxicity requires more attention and interest from researchers [57].

Conclusions
The results highlighted that identifying cyanobacteria isolates by combining morphological and molecular methods is still challenging.Furthermore, for the first time in this reservoir, a potentially toxic genotype from picocyanobacteria was described; this group of cyanobacteria is often overlooked, although its toxicity is of great importance.Thus, water treatment methods in this ecosystem must consider the presence of picocyanobacteria strain.This adaptation entails the implementation of pre-treatment techniques that can selectively eradicate picocyanobacteria while preserving the integrity of their cellular structure and therefore, the release of ovoid cyanotoxins.In addition, it is vitally important to conduct in-depth research on the function of picocyanobacteria in aquatic ecosystems.
represent their photomicrographs.

Figure 4 .
Figure 4. Phylogenetic relationships of Cheffia Reservoir cyanobacteria isolates with Genbank cyanobacteria strains based on 16S rRNA sequences.Escherichia coli (J01859) was used as the outgroup.Isolates from this study are underlined.A-E indicate the clusters; I, II indicate the sub-clusters.

Figure 4 .
Figure 4. Phylogenetic relationships of Cheffia Reservoir cyanobacteria isolates with Genbank cyanobacteria strains based on 16S rRNA sequences.coli (J01859) was used as the outgroup.Isolates from this study are underlined.A-E indicate the clusters; I, II indicate the sub-clusters.

Table 1 .
Primer sets used for PCR.

Table 3 .
Morphological characteristics of the colonial cyanobacterial strains isolated from the Cheffia Reservoir.
M. flos-aquae CR13 Spherical colonies, with irregular margins, microscopic to macroscopic, free-floating, compact, or clathrate, with densely irregularly arranged cells gathered in small agglomerations Mucilage colorless, slightly distant from cell clusters, and delimited by slightly refractive outline Cells spherical or hemispherical after division, with individual thick envelopes.Cell content appears granular, olive green, or brownish, with aerotopes 3.98-5.77

Table 4 .
16S rRNA gene-sequence-based identity (%) between the cyanobacterial isolates from the Cheffia Reservoir and their closest match available in Genbank (NCBI).